RNA-seq based SNPs in some agronomically important oleiferous lines of Brassica rapaand their use for genome-wide linkage mapping and specific-region fine mapping

Abstract

BackgroundBrassica rapa (AA) contains very diverse forms which include oleiferous types and many vegetable types. Genome sequence of B. rapa line Chiifu (ssp. pekinensis), a leafy vegetable type, was published in 2011. Using this knowledge, it is important to develop genomic resources for the oleiferous types of B. rapa. This will allow more involved molecular mapping, in-depth study of molecular mechanisms underlying important agronomic traits and introgression of traits from B. rapa to major oilseed crops - B. juncea (AABB) and B. napus (AACC). The study explores the availability of SNPs in RNA-seq generated contigs of three oleiferous lines of B. rapa - Candle (ssp. oleifera, turnip rape), YSPB-24 and Tetra (ssp. trilocularis, Yellow sarson) and their use in genome-wide linkage mapping and specific-region fine mapping using a RIL population between Chiifu and Tetra.ResultsRNA-seq was carried out on the RNA isolated from young inflorescences containing unopened floral buds, floral axis and small leaves, using Illumina paired-end sequencing technology. Sequence assembly was carried out using the Velvet de-novo programme and the assembled contigs were organised against Chiifu gene models, available in the BRAD-CDS database. RNA-seq confirmed the presence of more than 17,000 single-copy gene models described in the BRAD database. The assembled contigs and the BRAD gene models were analyzed for the presence of SSRs and SNPs. While the number of SSRs was limited, more than 0.2 million SNPs were observed between Chiifu and the three oleiferous lines. Assays for SNPs were designed using KASPar technology and tested on a F7-RIL population derived from a Chiifu x Tetra cross. The design of the SNP assays were based on three considerations - the 50 bp flanking region of the SNPs should be strictly similar, the SNP should have a read-depth of ≥7 and no exon/intron junction should be present within the 101 bp target region. Using these criteria, a total of 640 markers (580 for genome-wide mapping and 60 for specific-region mapping) marking as many genes were tested for mapping. Out of 640 markers that were tested, 594 markers could be mapped unambiguously which included 542 markers for genome-wide mapping and 42 markers for fine mapping of the tet-o locus that is involved with the trait tetralocular ovary in the line Tetra.ConclusionA large number of SNPs and PSVs are present in the transcriptome of B. rapa lines for genome-wide linkage mapping and specific-region fine mapping. Criteria used for SNP identification delivered markers, more than 93% of which could be successfully mapped to the F7–RIL population of Chiifu x Tetra cross.

RNA-seq based SNPs for mapping in Brassica juncea (AABB): synteny analysis between the two constituent genomes A (from B. rapa) and B (from B. nigra) shows highly divergent gene block arrangement and unique block fragmentation patterns

“…Equal amounts of RNA, 6 μg from each sample, were pooled and used for cDNA library preparation following the method described for RNA-seq of B. rapa lines [22]. …”

SNP diversity within and amongBrassica rapaaccessions reveals no geographic differentiation

“…Thereafter, genomes from two other morphotypes, turnip and oiltype, have been sequenced (Lin et al 2014). Nowadays, several studies have been published which describe the use of Infinium SNP array (Liu et al 2013;Raman et al 2014), or the development and use of novel SNP markersmainly for linkage mappingin Brassica A genome (Li et al 2009;Park et al 2010;Delourme et al 2013;Paritosh et al 2013;Devisetty et al 2014;Chung et al 2014;Zhang et al 2014;Song et al 2015). However, genetic diversity in oilseed B.rapa has not been studied with SNPs.…”

An RNA‐Seq‐based reference transcriptome for Citrus

“…Utilization of RNA-seq requires much less sequence depth to identify the majority of the variants in medium to highly expressed transcripts relative to whole genome sequencing. This advantage has been used, for example, to explore genetic variability in a large number of populations and varieties from Zea mays (Hansey et al, 2012) or Brassica rapa (Devisetty et al, 2014;Paritosh et al, 2013). Validation of SNPs by different methods showed rates higher than 90% indicating the accuracy of this method (Devisetty et al, 2014;Hansey et al, 2012;Scaglione et al, 2012).…”